IL-21-Mediated Generation of Human Regulatory B Cells Occurs Independently of CD40 Signaling

Recently, we and others found that B cells differentiate into regulatory B cells (B_reg) in response to interleukin (IL-)21. Of note, the key characteristic of human IL-21-induced B_reg is expression of the serine protease granzyme B (GrB), whereas murine B_reg, which require both IL-21 and CD40 ligand (CD40L) for their induction, predominantly express the immunosuppressive cytokine IL-10. Using two different disease models and various immunological methods, we further characterized the conditions leading to B_reg differentiation in humans. Here, we demonstrate that in humans CD40L determines whether IL-21 induces differentiation of B cells into plasma cells (CD40L presence) or into GrB⁺ B_reg (CD40L absence), which can directly control T cell proliferation by GrB-dependent degradation of the T cell receptor z-chain. Furthermore, we show that GrB⁺ B_reg are circulating at high frequencies in the peripheral blood of untreated, highly viremic HIV patients, but not in healthy subjects. Of note, HIV-infected CD4⁺ T cells express IL-21, but not CD40L, and induce a GrB⁺ regulatory phenotype in healthy third party B cells in vitro. Consequently, addition of CD40L multimers can compensate for this insufficient T helper cell function, resulting in increased plasma cell/B_reg ratios. Moreover, we investigated a patient with a congenital defect of Nuclear-Factor-kappa-B-Essential-Modulator (NEMO), which is essential for normal CD40 signaling. Even in the presence of viral infections, when CD4⁺ T helper cells from such patients are highly activated with strong expression of IL-21, they are not able to establish sufficient antibody responses. Instead, we found this patient to almost exclusively harbor B cells with a regulatory phenotype including high basal levels of GrB. When untreated NEMO B cells were co-cultured with allogeneic T cells from a healthy third party donor, these T cells failed to proliferate and to survive in response to a 6-day stimulation with anti-CD3/CD28 antibodies, an effect not observed with B cells from healthy donors. Since NEMO B cells lack normal CD40 signaling, our findings unequivocally demonstrate that in contrast to murine B_reg IL-21-dependent induction of human B_reg can occur in a CD40-independent fashion.